The SCF ubiquitin ligase pathway employs cullin proteins to assemble substrate specificity modules and ubiquitin conjugating enzymes, thereby promoting substrate ubiquitination. Our recent work has revealed that cullin-3 (Cul3) binds a large family of BTB-domain proteins in a manner analogous to the Skpl/F-box complex, which binds Cull. We hypothesize that BTB proteins function as substrate specific adaptors for Cul3. Evidence for this hypothesis comes from our finding that the C. elegans BTB protein Mel-26 interacts with its genetically defined target Mei-1 through its MATH domain. Elimination of Mei-1 protein that accompanies exit from meiosis depends upon Mel-26, Cul-3, and the cullin activator Nedd8, suggesting that the Cul3/Mel-26 complex functions as a ubiquitin ligase to facilitate Mei-1 destruction during this cell cycle transition. In addition, we have recently found that Keap1, which interacts through its BTB domain with Cul3 and through its kelch domain with the transcription factor Nrf2, is required for degradation of Nrf2 in vivo and ubiqutination of Nrf2 in vitro. Here, we seek to further establish the role of BTB proteins in targeted ubiquitintation. In Aim 1, we will elucidate the biochemical requirements for target ubiquitination by two Cul3-based ubiquitin ligases, Mel-26/Cul3 and Keap1/Cul3, and will examine the role of BTB-protein dimerization in ubiquitination activity in vitro and protein turnover in vivo. In aim 2, we will extend our structural analysis of E3s by examining the architecture of the Cul-3/Mel-26/Mei-1 complex through crystallographic and biochemical studies. In aim 3, we will employ newly developed RNAi libraries against BTB proteins to define the role of Cul3/BTB complexes in cell division and monomeric cyclin E turnover. In addition, novel approaches for the identification of ubiquitination targets employing loss-of-function genetics and applicable to genome-wide screens will be developed in the context of the BTB/Cul3 pathway. These experiments will: confirm and extend the hypothesis that BTB proteins function as substrate specific adaptors of Cul3, will reveal the structural basis for Cul-3 and substrate recognition by the Mel-26 BTB protein, and will apply the power of functional genomic approaches to the problem of ubiquitin ligase function and target identification in human cells.